Stimulated Emission Depletion (STED) imaging or microscopy can allow imaging with resolution much smaller than the wavelength of light used. STED imaging generally employs a pair of light beams, i.e., an excitation beam and a depletion beam, which can be simultaneously projected onto a spot on an object containing a fluorescent substance (e.g., containing proteins that are tagged with a fluorescent marker). The excitation beam has a frequency that excites the fluorescent substance so that presence of the fluorescent substance can be identified from emission of fluorescent light. In particular, the excitation beam excites atoms or molecules of the fluorescent substance to a higher energy state, from which molecules can decay and emit light of a characteristic wavelength, which is generally longer than the wavelength of the excitation beam. The excitation beam by itself can be tightly focused onto the target spot, so that the amount of fluorescent substance in the target spot can be determined from the intensity of fluorescent light emitted if the excitation beam is used by itself. However, the minimum area illuminated by the focused excitation beam is diffraction limited and generally has a minimum dimension on the order of the wavelength of the excitation beam, which limits the resolution of an image created solely using the excitation beam and fluorescence detection.
The depletion beam for STED imaging has a wavelength selected to stimulate specific emissions from the fluorescent substance to thereby deplete the population of the fluorescent substance in the excited state that provides the measured fluorescence. As a result, an area of an object illuminated with a sufficient intensity of the depletion beam will not fluoresce at the target frequency when simultaneously exposed to the excitation beam, even if the fluorescent substance is present in the illuminated area. STED imaging can achieve a small spot size and therefore high (e.g., sub-wavelength) resolution using a doughnut-shaped intensity profile for the depletion beam. If both the excitation beam and the doughnut-shaped depletion beams illuminate an area of an object, the measured fluorescence is only significant where the intensity of the excitation beam is high relative to the depletion beam (e.g., in the hole of the doughnut-shaped intensity profile of the depletion beam). The area in which the excitation beam is sufficiently intense relative to the depletion beam can be made much smaller than the area of a diffraction-limited Guassian beam profile. Accordingly, STED imaging has achieved image resolutions down to about 20 nm using light with wavelength of about 600 nm.
STED imaging needs an optical system capable of producing a depletion beam with a doughnut-shaped beam profile. Such a beam profile can be achieved using a spiral zone plate, which applies to a beam a phase shift that varies linearly across a 2π range as a function of an angle around a central axis. Using current technology, a spiral zone plate generally uses plate thickness to create phase shifts and has a thickness that varies continuously with angle. These spiral zone plates can be difficult to fabricate using current semiconductor processing techniques.
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